RESUMEN
Apelin (Apln) is a myokine that regulates skeletal muscle plasticity and metabolism and declines during aging. Through a yeast one-hybrid transcription factor binding screen, we identified the TEA domain transcription factor 1 (Tead1) as a novel regulator of the Apln promoter. Single-cell analysis of regenerating muscle revealed that the apelin receptor (Aplnr) is enriched in endothelial cells, whereas Tead1 is enriched in myogenic cells. Knock-down of Tead1 stimulates Apln secretion from muscle cells in vitro and myofiber-specific overexpression of Tead1 suppresses Apln secretion in vivo. Apln secretion via Tead1 knock-down in muscle cells stimulates endothelial cell expansion via endothelial Aplnr. In vivo, Apln peptide supplementation enhances endothelial cell expansion while Tead1 muscle overexpression delays endothelial remodeling following muscle injury. Our work describes a novel paracrine crosstalk in which Apln secretion is controlled by Tead1 in myogenic cells and influences endothelial remodeling during muscle repair.
RESUMEN
AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Hepatocitos , Lípidos , Fenantrenos , Proteínas Quinasas Activadas por AMP/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Diabetes Mellitus Tipo 2 , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Metabolismo de los Lípidos , Lípidos/biosíntesis , Ratones , Fenantrenos/farmacología , FosforilaciónRESUMEN
AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proteínas Proto-Oncogénicas/fisiología , Serina-Treonina Quinasas TOR/fisiología , Proteínas Supresoras de Tumor/fisiología , Transporte Activo de Núcleo Celular , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Fosforilación , Ribonucleótidos/farmacología , Pez CebraRESUMEN
PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK) 16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been identified in human patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only a few cellular targets/substrates have been proposed with no clear functional significance. We and others recently showed that cyclin Y binds and activates PCTAIRE-1 via phosphorylation and 14-3-3 binding. In order to understand the physiological role that PCTAIRE-1 plays in brain, we have performed a chemical genetic screen in vitro using an engineered PCTAIRE-1/cyclin Y complex and mouse brain extracts. Our screen has identified potential PCTAIRE-1 substrates (AP2-Associated Kinase 1 (AAK1), dynamin 1, and synaptojanin 1) in brain that have been shown to regulate crucial steps of receptor endocytosis, and are involved in control of neuronal synaptic transmission. Furthermore, mass spectrometry and protein sequence analyses have identified potential PCTAIRE-1 regulated phosphorylation sites on AAK1 and we validated their PCTAIRE-1 dependence in a cellular study and/or brain tissue lysates. Our results shed light onto the missing link between PCTAIRE-1 regulation and proposed physiological functions, and provide a basis upon which to further study PCTAIRE-1 function in vivo and its potential role in neuronal/brain disorders.
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Encéfalo/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Dinamina I/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Encéfalo/citología , Células COS , Chlorocebus aethiops , Ciclinas/genética , Dinamina I/genética , Pruebas Genéticas , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Especificidad por SustratoRESUMEN
AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.
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Proteínas Quinasas Activadas por AMP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Animales , Hepatocitos/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Metabolismo de los Lípidos/genética , Espectrometría de Masas/métodos , Ratones Noqueados , Fosforilación , Especificidad por SustratoRESUMEN
To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.
Asunto(s)
Productos Biológicos/química , Trifolium/química , Proteínas Quinasas Activadas por AMP/metabolismo , Algoritmos , Cromatografía Líquida de Alta Presión , Genisteína/química , Estructura Molecular , Estándares de Referencia , SuizaRESUMEN
AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory ß (ß1 and ß2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 µM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Bencimidazoles/farmacología , Benzoatos/farmacología , Activadores de Enzimas/farmacología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Músculo Esquelético/efectos de los fármacos , Ribonucleótidos/farmacología , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Aminoimidazol Carboxamida/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Humanos , Técnicas In Vitro , Isoenzimas , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacosRESUMEN
PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3.
Asunto(s)
Proteínas 14-3-3/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Quinasas Ciclina-Dependientes/genética , Ciclinas/química , Ciclinas/genética , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Alineación de SecuenciaRESUMEN
AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Hepatocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Quinasas Activadas por AMP/análisis , Secuencia de Aminoácidos , Animales , Células CACO-2 , Células Cultivadas , Humanos , Masculino , Espectrometría de Masas , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/análisis , Datos de Secuencia Molecular , Fosforilación , ProteómicaRESUMEN
LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1-SIK pathway functions as a key gluconeogenic gatekeeper in the liver.
Asunto(s)
Gluconeogénesis/genética , Hiperglucemia/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Animales , Ayuno , Regulación de la Expresión Génica , Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hiperglucemia/metabolismo , Hiperglucemia/patología , Insulina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/farmacología , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, ß1, or ß2.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Activadores de Enzimas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Quinasas Activadas por AMP/química , Adenosina Monofosfato/farmacología , Secuencia de Aminoácidos , Animales , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/metabolismo , Esterificación/efectos de los fármacos , Ácidos Grasos/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Profármacos/metabolismo , Profármacos/farmacología , Subunidades de Proteína/agonistas , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Especificidad por SustratoRESUMEN
Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS-GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/enzimología , Glucosiltransferasas , Glucógeno Sintasa , Glicoproteínas , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema Libre de Células , Células Cultivadas , Cristalografía por Rayos X , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glucógeno/biosíntesis , Glucógeno/química , Glucógeno/genética , Glucógeno Sintasa/química , Glucógeno Sintasa/genética , Glucógeno Sintasa/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Ratones , Ratones Noqueados , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFßTrCP (Skp, Cullin and F-boxßTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to ßTrCP, the substrate-recognition subunit of the SCFßTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2-M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1ßMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1ßMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFßTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1ßMYPT1 phosphatase.
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Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Fosfatasa de Miosina de Cadena Ligera/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina-Proteína Ligasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Alineación de Secuencia , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Quinasa Tipo Polo 1RESUMEN
Precise homoeostasis of the intracellular concentration of Cl- is achieved via the co-ordinated activities of the Cl- influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+-K+ ion co-transporters), also promote inhibition of the KCCs (K+-Cl- co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl- influx, we propose that the targeting of WNK-SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl- extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.
Asunto(s)
Cloruros/metabolismo , Potasio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Simportadores/antagonistas & inhibidores , Simportadores/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Fosfopéptidos/metabolismo , Fosforilación , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Transducción de Señal , Simportadores de Cloruro de Sodio-Potasio/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Activation of AMPK provides beneficial outcomes in fighting against metabolic disorders such as insulin resistance and type 2 diabetes. Currently, there is no allosteric AMPK activator available for the treatment of metabolic diseases, and limited compounds are available to robustly stimulate cellular/tissue AMPK in a specific manner. Here we investigated whether simultaneous administration of two different pharmacological AMPK activators, which bind and act on different sites, would result in an additive or synergistic effect on AMPK and its downstream signaling and physiological events in intact cells. We observed that cotreating primary hepatocytes with the AMP mimetic 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) and a low dose (1 µM) of the allosteric activator A769662 produced a synergistic effect on AMPK Thr172 phosphorylation and catalytic activity, which was associated with a more profound increase/decrease in phosphorylation of downstream AMPK targets and inhibition of hepatic lipogenesis compared with single-compound treatment. Mechanistically, we found that cotreatment does not stimulate LKB1, upstream kinase for AMPK, but it protects against dephosphorylation of Thr172 phosphorylation by protein phosphatase PP2Cα in an additive manner in a cell-free assay. Collectively, we demonstrate that AICAR sensitizes the effect of A769662 and promotes AMPK activity and its downstream events. The study demonstrates the feasibility of promoting AMPK activity by using two activators with distinct modes of action in order to achieve a greater activation of AMPK and downstream signaling.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Activadores de Enzimas/farmacología , Hepatocitos/efectos de los fármacos , Hipoglucemiantes/farmacología , Mioblastos/efectos de los fármacos , Pironas/farmacología , Ribonucleótidos/farmacología , Tiofenos/farmacología , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Regulación Alostérica/efectos de los fármacos , Aminoimidazol Carboxamida/farmacología , Animales , Compuestos de Bifenilo , Línea Celular , Células Cultivadas , Glucosa/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Lipogénesis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mioblastos/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Subunidades de Proteína/agonistas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/toxicidad , Animales , Animales Modificados Genéticamente , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Mutación , Neuronas/citología , Neurotoxinas/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some ß-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the ß-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.
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Cromatografía de Afinidad/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Animales , Tampones (Química) , Dictyostelium/metabolismo , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Insectos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Saccharomyces cerevisiae/metabolismoRESUMEN
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
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Potencial de la Membrana Mitocondrial/fisiología , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Enfermedad de Parkinson/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Quinasas/genética , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/farmacología , Proteínas Recombinantes de Fusión/fisiología , Tribolium/enzimología , Tribolium/genéticaRESUMEN
Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Células Madre Embrionarias , Regulación de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutación , Fosforilación , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Simportadores de Cloruro de Sodio-Potasio/genética , Miembro 2 de la Familia de Transportadores de Soluto 12 , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.